As used here, and hereinafter, the expression "predetermined analyte" is defined as a compound either (a) that is capable of interacting with an oxidase enzyme to generate hydrogen peroxide or (b) that exhibits peroxidative activity. Similarly, as used here, and hereinafter, the expression "redox mediator" is defined as a compound, or compounds, capable of interacting with a predetermined analyte to generate molecular oxygen. The molecular oxygen, in turn, oxidizes an oxidizable dye to provide a detectable color change. The "redox mediator" can be a combination of an oxidase enzyme and peroxidase enzyme, or can be a hydroperoxide, depending upon the particular predetermined analyte of interest. Therefore, assays for a predetermined analyte, like an analyte capable of interacting with a suitable oxidase enzyme and peroxidase, such as glucose, or like a peroxidatively active substance, such as occult blood, i.e., hemoglobin, are based upon a chromogenic interaction, wherein the degree and intensity of a color transition of an oxidizable indicator dye are correlated to the concentration of the predetermined analyte in a test sample. Assays for a predetermined analyte are particularly useful in detecting and measuring low concentrations of analyte in body fluid samples such as blood, urine, feces or gastrointestinal contents.
For example, glucose is the sugar most commonly found in urine. The presence of detectable amounts of glucose in urine is known as glycosuria. Glycosuria can be a benign or a pathological condition, and the physician must distinguish between the two types.
Glycosuria can occur when blood glucose levels are normal because reabsorption of glucose in the . kidneys is below normal, thus permitting some glucose to spill into the urine. This is a benign condition, as is the occurrence of glycosuria after eating a heavy meal or in conjunction with emotional stress. However, diabetes mellitus is a pathological state and is the chief cause of glycosuria. Indications of diabetes mellitus include a marked elevation of blood glucose and an increase in urine volume. The urine glucose content of a diabetic individual can be as high as 10%, with a content of 2% to 5% commonly being found.
Various assays are available to test urine for glucose. The most commonly used assay is an enzymatic test based on an interaction between glucose oxidase and glucose. The enzymatic glucose oxidase test for glucose, as applied to urine, is specific for glucose. Other sugars, such as lactose, fructose, galactose and pentose, are not substrates for the glucose oxidase enzyme, and, therefore, are not detected or measured.
In a standard assay of a test sample for glucose, glucose oxidase, in the presence of oxygen, first converts the glucose in the test sample to gluconic acid and hydrogen peroxide. Then, the peroxidase enzyme, also present in the assay, catalyzes an interaction between the hydrogen peroxide and an oxidizable dye compound, like o-tolidine. The dye compound, usually essentially colorless in its reduced state, undergoes a color transition upon oxidation, such as to a blue color for o-tolidine, by the peroxidase-catalyzed interaction with hydrogen peroxide. The degree and intensity of the color transition are directly proportional to the amount of hydrogen peroxide generated by the glucose conversion. Then, the amount of hydrogen peroxide generated by the glucose conversion is correlated to the original concentration of glucose in the urine sample. In practice, a test strip is dipped into the urine sample, and the resulting color transition of the test strip is compared to a color chart ranging from colorless, indicating less than 0.1% concentration of glucose, to blue, indicating a 2.0% or greater concentration in the urine.
Peroxidase is an enzyme that catalyzes the oxidation of various compounds, such as phenols and amines, by peroxides. In addition, particular compounds have been termed pseudoperoxidases because these compounds behave in a manner similar to the peroxidase enzyme. Accordingly, pseudoperoxidases liberate oxygen from hydroperoxides, and transfer the oxygen to certain acceptor compounds. Therefore, in general, the pseudoperoxidases are enzyme-like in that they catalyze, or otherwise participate in, interactions between peroxides and oxidizable compounds, like oxidizable dye compounds. The pseudoperoxidases also are termed peroxidatively active substances, and include hemoglobin and its derivatives.
Therefore, a peroxidatively active substance, like hemoglobin, a hemoglobin derivative, an erythrocyte, myoglobin or a combination thereof, catalyzes an interaction between a hydroperoxide and an oxidizable dye. In such interactions, the peroxidatively active substance imitates the peroxidase enzyme, and catalyzes or otherwise participates in an interaction between the oxidizable dye and the hydroperoxide. The oxygen liberated from a hydroperoxide by a peroxidatively active substance is transferred to an oxidizable dye. The resulting interaction provides a detectable response, such as a color transition, wherein the intensity and degree of the response are indicative of the presence or the concentration of the peroxidatively active substance.
For example, a low concentration of blood in the urine is termed "occult blood." Occult blood is detected by assaying for the peroxidatively active compound hemoglobin. Although occult blood in urine, feces or vomit usually is not visible to the naked eye, the detection of occult blood is important in the diagnosis of hemorrhages in the stomach, intestines and urinary tract. The hemorrhages are caused, for example, by tumors, ulcers or inflammations of the organ in question. Presently, most methods of determining the presence of occult blood in a test sample are based upon the pseudoperoxidase activity of hemoglobin or myoglobin.
The presence of blood in urine also is an indication of damage to the kidney or urinary tract. Normally, detectable amounts of occult blood are not present in urine, even with very sensitive chemical methods. The presence of blood in urine or feces is a symptom of a variety of abnormal conditions, including cancer. The presence of blood in urine, as indicated by a positive test for occult blood, often indicates bleeding in the urinary tract. Free hemoglobin is present in the urine because of renal disorders, infectious diseases, neoplasms, or traumas affecting part of the urinary tract. Free hemoglobin in the urine also can indicate a transfusion reaction, hemolytic anemia, or paroxysmal hemoglobinuria, or can appear from various poisonings or following severe burns.
Therefore, accurate and sensitive assays of blood, urine and other test samples for various predetermined analytes must be available for both laboratory and home use. The assays should provide an accurate detection and measurement of the predetermined analyte such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the assay method could be utilized in a dip-and-read format for the easy and economical, qualitative or quantitative determination of a predetermined analyte in blood, urine or other test samples.
Furthermore, any method of assaying for a particular predetermined analyte in blood, urine or other test samples must yield accurate, trustworthy and reproducible results by utilizing an indicator reagent composition that undergoes a color transition as a result of an interaction with the predetermined analyte, and not as a result of a competing chemical or physical interaction, such as a preferential interaction with a test sample component, like ascorbate, other than the predetermined analyte. Moreover, it would be advantageous if the assay method for the predetermined analyte is suitable for use in dry phase reagent strips for the rapid, economical and accurate determination of the predetermined analyte in blood, urine or other test samples. Additionally, the method and composition utilized in the assay for the predetermined analyte should not adversely affect or interfere with the other test reagent pads that are present on multideterminant reagent strips.
In order to determine if a body fluid of an individual includes a clinically-significant amount of a predetermined analyte, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive detection assays for predetermined analytes, like glucose and occult blood, have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of a predetermined analyte in a test sample, the methods based on dip-and-read dry phase test strips have proven especially useful because dry phase test strip methods are readily automated and provide reproducible and accurate results.
Some test strips used in assays for a predetermined analyte have a single test area consisting of a small square pad of a suitable carrier matrix impregnated with an indicator reagent composition capable of interacting with the predetermined analyte and undergoing a detectable or measurable change, such as a color transition. Other test strips are multideterminant reagent strips that include one test area for the assay of a particular predetermined analyte as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other clinically-important constituents present in the test sample. For both types of colorimetric test strips, the assay for a predetermined analyte in the test sample, such as blood or urine, is performed simply by dipping the colorimetric test strip into the test sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle. For example, glucose and occult blood tests usually are included on multideterminant reagent strips to screen urine samples during routine physical examinations because it is important to detect excess amounts of these urinary constituents early.
The test strip method is the simplest and most direct assay for the presence of a predetermined analyte. In an assay for glucose, the test area incorporates an oxidizable indicator dye, like 3,3',5,5'-tetramethylbenzidine; glucose oxidase; and peroxidase. In an assay for occult blood, the test area incorporates an oxidizable indicator dye and a hydroperoxide. In either assay, the test area undergoes a color transition in response to an interaction between the predetermined analyte present in the test sample and the glucose oxidase-peroxidase couple, or the hydroperoxide, to oxidize the tetramethylbenzidine. In accordance with the above-described method, an individual can readily determine, visually, the concentration of the predetermined analyte in a urine sample by comparing the color of the test strip to a color chart shortly after the test strip is dipped into the test sample.
However, ascorbic acid or ascorbate ion, when present in a test sample, seriously interferes in the above-described oxidation-reduction assay method for a predetermined analyte. The most common form of ascorbic acid typically is referred to as Vitamin C. This vitamin is a vital nutrient and is found in many naturally-occurring foods, such as fruits and vegetables. Vitamin C also can be synthesized and is therefore available as a food additive or in tablet form. The health benefits of Vitamin C have been known for some time, as a result, Vitamin C is a relatively popular nutrient. Therefore, Vitamin C is a popular food additive and a popular component of vitamin pills and the like.
However, the human body generally absorbs Vitamin C only to the extent necessary to meet short term needs. The vitamin usually is not stored within the body, and excess Vitamin C typically is disposed of through the urinary system. As a result, Vitamin C commonly is present in urine samples undergoing clinical assays.
Ascorbic acid is a reducing agent that can interfere in clinical assays by reducing the oxidized, colored form of an indicator dye to the reduced, colorless form of the dye. However, ascorbic acid can be oxidized. Therefore, if the ascorbic acid is oxidized before it can interact with the oxidized indicator dye, the ascorbic acid cannot act as a reducing agent and accordingly cannot interfere with the assay for a predetermined analyte.
Either in the literature or during in-house screening studies, it has been found that including certain metal ion complexes in the indicator reagent composition helps eliminate the ascorbate interference problem. However, a metal ion complex also can oxidize the dye chemically or can demonstrate peroxidase activity, and behave similarly to peroxidase enzyme or to a pseudoperoxidase, to catalyze the color-forming reaction between hydrogen peroxide or a hydroperoxide and an oxidizable dye. Accordingly, although a metal ion complex eliminates ascorbate interference, the metal ion complex may produce false positive assays.
Investigators have found that particular ferric ion complexes substantially reduce the false positive assay results attributed to some metal ion complexes used to eliminate ascorbate interference. Ascorbic acid interferences are eliminated because ascorbic acid is oxidized by ferric ion or by ferric ion complexes. Representative publications illustrating the ferric ion oxidation of ascorbic acid include:
E. Pelizetti et al., "Kinetics and Mechanism of the Oxidation of Ascorbic Acid by Tris(1,10-phenanthroline)iron(III) and Its Derivatives in Aqueous Acidic Perchlorate Media", Inorg. Chem., 15, pp. 2898-2900 (1976), wherein ascorbic acid was reacted with tris(1,10-phenanthroline)iron(III) in aqueous perchlorate over a pH range of one to 3.5, with the rate of oxidation decreasing with increasing pH;
L. S. Vann, "A Rapid Micro Method for Determination of Ascorbic Acid in Urine by Ferric Reduction", Clin. Chem., 11, pp. 979-985 (1965);
M. M. T. Khan et al., "The Kinetics of the Reaction of Iron (III) Chelates of Aminopolycarboxylic Acids with Ascorbic Acid", J. Am. Chem. Soc., 90, pp. 3386-3389 (1968) and M. M. T. Khan et al., J. Am. Chem. Soc., 89, p. 7104 (1967), wherein the kinetics of ascorbic acid oxidation in the presence of ferric and cupric chelates in the pH range of 1.8 to 3.45 is discussed;
G. S. Laurence et al., "The Detection of a Complex Intermediate in the Oxidation of Ascorbic Acid by Ferric Ion", J. Chem. Soc. Dalton Trans., pp. 1667-1670 (1972);
W. C. Butts et al., "Centrifugal Analyzer Determination of Ascorbate in Serum and Urine with Fe.sup.3+ /Ferrozine", Clin. Chem., 21, pp. 1493-1497 (1975);
L. Pekkarinen, "The Mechanism of the Autoxidation of Ascorbic Acid Catalyzed by Iron Salts in Citric Acid Solution", Finn. Chem. Lett., pp. 233-236 (1974);
M. Kimura et al., "Kinetics and Mechanism of the Oxidation of L-Ascorbic Acid by Tris-(oxalato) Cobaltate(III) and Tris(1,10-phenanthroline)Iron(III) Complexes in Aqueous Solution", J. Chem. Soc. Dalton Trans., pp. 423-427 (1982); and
A. E. Martell, "Chelates of Ascorbic Acid, Formation and Catalytic Properties", Ascorbic Acid Chemistry, Metabolism and Uses, Chapter 7, P. A. Seib and B. M. Tolbert eds., Adv. in Chem. Series, ACS, Wash., D.C., pp. 153-178, (1982).
However, the ferric ion-based oxidation of ascorbic acid described above presents a definite disadvantage when used to eliminate ascorbic acid interference in an oxidase-peroxidase coupled reaction to assay for a predetermined analyte. In the oxidation of ascorbic acid, ferric ion is reduced to ferrous ion. Ferrous ion is a good reducing agent and can reduce an oxidized indicator dye, like tetramethylbenzidine (TMB), from its colored (oxidized) form to its colorless (reduced) form. Therefore, although the ferric ion eliminates the primary ascorbic acid interference, the ferrous ion then produces a secondary interfering affect that can result in an erroneously low assay result.
Accordingly, it would be extremely advantageous to provide a simple, accurate and trustworthy method of assaying a test sample for low levels of a predetermined analyte without the primary or secondary interfering affects attributed to ascorbic acid. Present day test strips for a predetermined analyte incorporate an indicator reagent composition including a suitable oxidase enzyme; peroxidase enzyme; and a metal ion complex to reduce primary ascorbate interferences. Although present day test strips used to assay for a predetermined analyte are stable and sensitive, present day test strips still need improvement in the area of sensitivity. Therefore, it would be a significant advance in the art of diagnostic assays if test strips were even more sensitive to a predetermined analyte in a test sample. It was towards achieving improvements in ascorbate resistance and sensitivity that the investigations resulting in the composition, device and method of the present invention were directed.
Surprisingly and unexpectedly, the composition and method of the present invention eliminate primary ascorbate interference and secondary interferences attributed to the metal ion or metal ion complex by including a cooxidant selected from the group consisting of bromate ion, chlorate ion, perchlorate ion, chromate ion, organic oxidants like a peroxide, a hydroperoxide or a N-halo compound, and combinations thereof, in the indicator reagent composition. To achieve the full advantage of the present invention, bromate ion is included in the indicator reagent composition as the cooxidant. It has been found that the cooxidant oxidizes the reduced form of the metal ion, e.g., ferrous ion, to the oxidized form, e.g., ferric ion, such that the reduced form of the metal ion is unavailable to interact with the oxidized form of the indicator dye. Therefore, the problem of decreased assay sensitivity attributed to a metal ion or a metal ion complex included in the indicator reagent composition is overcome.
Accordingly, a quantitative assay for a predetermined analyte can be performed by laboratory personnel to yield immediate and trustworthy test results by providing a more accurate assay method in an easy-to-use form, such as a dip-and-read test strip. In addition, the test strip method can be performed by an individual at home to more precisely monitor the level of a predetermined analyte, like glucose or occult blood, in a test sample, like blood or urine, or to monitor the success of the medical treatment the individual is undergoing.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy assay for a predetermined analyte by utilizing a test strip that includes a test pad comprising a suitable carrier matrix incorporating an indicator reagent composition of the present invention, said indicator reagent composition comprising an indicator dye, a redox mediator, a metal ion complex and a cooxidant. If the predetermined analyte, like glucose, is capable of interacting with an oxidase enzyme, the redox mediator comprises a suitable oxidase enzyme and a peroxidase enzyme. If the predetermined analyte, like occult blood, demonstrates peroxidative activity, the redox mediator comprises a hydroperoxide. The cooxidant is selected from the group consisting of bromate ion, chlorate ion, perchlorate ion, chromate ion, an organic oxidant like a peroxide, a hydroperoxide or a N-halo compound, and combinations thereof.
Prior to the present invention, no known method of assaying a test sample for a predetermined analyte in an oxidation-reduction coupled reaction included an indicator reagent composition comprising an indicator dye; a redox mediator; a metal ion complex; and a cooxidant selected from the group consisting of chlorate ion, bromate ion, perchlorate ion, chromate ion, an organic oxidant like a peroxide, a hydroperoxide or a N-halo compound, and combinations thereof. Although dry phase test strips including an indicator dye, a redox mediator and a metal ion complex have been used previously, dry phase test strips incorporating such compositions demonstrated a tendency to provide erroneous assay results because of secondary interferences attributed to the metal ion of the metal ion complex. Accordingly, such erroneous assays decreased the sensitivity of the test strip to the predetermined analyte in the test sample. In contrast, the indicator reagent composition of the present invention essentially eliminates both the primary ascorbate interference and, surprisingly and unexpectedly, the secondary interferences attributed to the metal ion complex. Consequently, the improved indicator reagent composition enhances the sensitivity of the assay, thereby providing a more accurate and trustworthy assay for a predetermined analyte by an oxidation-reduction based chemistry.
Several other attempts at achieving the above-mentioned goals of increased assay sensitivity and decreased ascorbate interference are found in prior patents and publications. For example, with regard to ascorbate interferences in glucose assays, methods have ranged from filtering the ascorbate from the test sample before the test sample contacts the test reagents to using an enzyme that interacts with the ascorbate. Accordingly, Canadian Patent No. 844,564 to Dahlquist disclosed a test device for glucose assays that includes a porous area to receive the test sample. The sample-receiving area does not include assay reagents, but comprises an ion exchange material that absorbs the ascorbate present in the test sample.
U.S. Pat. No. 4,168,205 to Danninger et al. described incorporating ascorbate oxidase into the test reagent formulation to enzymatically oxidize the ascorbate present in the sample to dehydroascorbate, a compound that does not adversely affect the assay. Japanese Patent Publication No. 55757 (1983) to Fuji Zoki Seiyaku K.K. disclosed pretreating a test sample with a metal chelate of a ligand, such as ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid, to eliminate ascorbate, then assaying the test sample for cholesterol, glucose or uric acid.
Ku, in U.S. Pat. No. 3,411,887, described the elimination of ascorbate interference with reagent compositions that rely on enzymatic oxidizing substances, such as glucose oxidase, by using an ascorbate "trapping system." The "trapping system" utilizes a heavy metal ion that has an oxidation-reduction potential falling between a redox indicator dye and ascorbate. Suitable heavy metal compounds cited as examples include cobalt, iron, mercury and nickel. Another publication disclosing the complexing and oxidation of ascorbate using cobalt is G. Bragagnolo, Ann. Chim. Applicata., 31, pp. 350-368 (1941), teaching that solutions of ascorbic acid were oxidized by air in the presence of cobalt metal. Also, similar activity has been reported for Co(NH.sub.3).sub.6 Cl.sub.3 by T. Iwasaki, Journal of the Chemical Society of Japan, 63, pp. 820-826 (1942).
U.S. Pat. No. 4,310,626 to Burkhardt et al. described the use of ammonium cobalt(III) complexes to reduce ascorbate interference in assays for peroxidatively active substances. Burkhardt et al. disclose compositions comprising an organic hydroperoxide and a suitable indicator, such as 3,3',5,5'-tetramethylbenzidine, together with ammonium cobalt(III) complexes, such as Co(NH.sub.3).sub.6 Cl.sub.3.
Copper ions also have been used to eliminate ascorbate interference from assays. For example, I. Pecht, et al., in "The Copper-Poly-L-Histidine Complex: I. The Environmental Effect of the Polyelectrolyte on the Oxidase Activity of Copper Ions", J. Am. Chem. Soc., 89:1587 (1968), disclosed that ascorbate can be oxidized by oxygen and a copper catalyst. N. A. Vengerova et al. in the publication, "The Ascorbate-Oxidase Activity of the Cu.sup.+2 -Poly-4-Vinylpyridine Complex Alkylated with Bromoacetic Acid", Vysokomol. soyed., A 13, No. 11, pp. 2509-2517 (1971) (translated by K. A. Allen), disclosed a method of synthesizing carboxymethyl derivatives of poly-4-vinylpyridine and taught that a Cu(II) polymer complex increases ascorbate oxidizing activity relative to copper ions alone. Other references relating to Cu(II) oxidation of ascorbate include: Z. Sun et al., "Studies on Functional Latices: Catalytic Effects of Histamine-Containing Polymer-latex-copper (II) Complex on the Oxidation of Ascorbic Acid", Macromolecules, 19:984-987 (1986); and Y. I. Skurlator et al., "The Mechanism of Ascorbic Acid Oxidation by Cu(II)-Poly-4-Vinylpyridine Complexes", European Polymer Journal, 15:811-815 (1979).
U.S. Pat. No. 4,288,541 to Magers et al. disclosed the use of mercuric ion complexes, such as mercuric sarcosinate, to impart ascorbate resistance to a glucose oxidase-based assay for glucose. In addition to the above patents and publications, the problem of ascorbate interference in glucose assays is discussed in:
H. Gifford, et al., J. Amer. Med. Assoc., 178, pp. 149-150 (1961);
P. O'Gorman, et al., Brit. Med. J., pp. 603-606 (1960);
R. Brandt, et al., Clin. Chem. Acta., 51, pp. 103-104 (1974); and
R. Brandt, et al., Am. J. Clin. Pathol., 68, pp. 592-594 (1977).
Other methods of eliminating ascorbate interference in analytical determinations of predetermined analyte include, for example, West German Patent No. 29 07 628, directed to a wet phase urinalysis, whereby a urine sample is pretreated with an oxidant to remove ascorbate prior to the assay. The oxidants disclosed as useful are sodium iodate, sodium periodate, calcium hydrochlorite, potassium triiodide, sodium hydrochlorite, chloroamine and bromosuccinimide. Also, European Patent Application 0037056 described the use of iodate in diagnostic methods to avoid interference by reducing agents, including ascorbic acid.
U.S. Pat. No. 4,587,220, to Mayambala-Mwanika et al., disclosed the use of a chelated ferric ion to eliminate ascorbate interference in an assay for a peroxidatively active substance. Mayambala-Mwanika disclosed that a ferric chelate, like the ferric chelate of N-(2-hydroxyethyl)ethylenediaminetriacetic acid (Fe-HEDTA), eliminated ascorbate interference and did not produce a false positive test for a peroxidatively active compound.
Ismail et al., in U.S. Pat. No. 4,755,472, disclosed a stable test pad to assay for a peroxidatively active substance that includes a carrier matrix impregnated with 1,4-diisopropylbenzene dihydroperoxide and a benzidine indicator in a molar ratio of hydroperoxide to indicator of from about 0.9 to 3.0. A ferric chelate also can be included to provide ascorbate resistance. The test pad was stable during storage and does not lead to false positive tests on other test pads present on a multideterminant test strip, such as glucose test pad based on a peroxidase/potassium iodide indicator.
In contrast to the prior art, and in contrast to the presently-available commercial test strips, the composition, method and device of the present invention demonstrate increased sensitivity in an assay to detect or measure the concentration of a predetermined analyte in a test sample, wherein the predetermined analyte either is capable of interacting with an oxidase enzyme or demonstrates peroxidase activity. The method of the present invention utilizes an indicator reagent composition that a) effectively eliminates primary ascorbate interferences by including a metal ion complex, and b) effectively eliminates secondary interferences attributed to the metal ion complex by including a cooxidant selected from the group consisting of bromate ion, chlorate ion, perchlorate ion, chromate ion, organic oxidants like a peroxide, a hydroperoxide or a N-halo compound, and combinations thereof.
For example, bromate ion is used in analytical chemistry as a titrimetric reagent. In acidic media, bromate ion is almost as powerful an oxidizing agent as permanganate ion. In the presence of strong reducing agents, bromate ion (BrO.sub.3.sup.-) is reduced to bromide ion (Br.sup.-). However, bromate ion is capable of oxidizing bromide ion to bromine (Br.sub.2) as demonstrated in the following equations (1) and (2): EQU BrO.sub.3.sup.- +6H.sup.+ +6e.sup.- .fwdarw.Br.sup.- +3H.sub.2 O(1) EQU BrO.sub.3.sup.- +5Br.sup.- +6H.sup.+ .fwdarw.3Br.sub.2 +2H.sub.2 O(2)
I. M. Kolthoff et al., in Volumetric Analysis, Vol. III, Titration Methods: Oxidation-Reduction Reactions, Chapter XII, Interscience Publishers, N.Y., N.Y. (1971), teach that bromide ion is formed in the first step of the reaction (Eq. 1), then the bromide ion reacts with excess bromate to yield free bromine (Eq. 2) in a pH-dependent reaction sequence. From Eqs. (1) and (2), it is demonstrated that acidic conditions are necessary for the reaction to proceed. Similarly, it is known that ; chlorate ion (ClO.sub.3.sup.-) does not demonstrate oxidizing properties in a neutral or alkaline solution, whereas chlorate ion demonstrates strong oxidizing properties in acidic solutions due to the presence of chloric acid (HClO.sub.3).
The bromate ion oxidation of reduced metal ions also has been described. For example, the bromate ion oxidation of ferrous ion, i.e., iron(II), has been described in:
J. P. Birk, "Mechanism of the Bromate Ion Oxidation of Aquoiron(II)", Inorg. Chem., 12, pp. 2468-2472 (1973);
J. P. Birk et al., "Mechanism of the Reduction of Bromate Ion by Cyano(bipyridyl) Iron(II) Complexes", Inorg. Chem., 17, pp. 1186-1191 (1978); and
S. G. Kozub, "Kinetics and Mechanisms of the Bromate Oxidations of Substitution--Inert Iron(II) Complexes in Acidic Aqueous Solution," Ph.D. Dissertation, Xerox University Microfilms, Ann Arbor, (1975).
In each above-identified publication, the reaction between bromate ion and ferrous ion was studied at very acidic conditions in perchloric acid. Furthermore, it is known that bromate ion does not oxidize ascorbic acid at an appreciable rate in the essentially neutral pH range of from 5 to 7.
U.S. patent application Ser. No. 337,620, filing date Apr. 13, 1989 and commonly assigned to the assignee of the present invention, describes the elimination of ascorbate interference from clinical assays by utilizing a copper(II) complex including a water-soluble polymer. In addition, a cooxidant is included to oxidize the copper(I) ion back to copper(II) after the interaction with ascorbate. The cooxidant can be an organic oxidant, like a peroxide or a N-halo compound, or an inorganic oxidant, like chromate, mercuric ion, thallium(III) ion, ceric(IV) ion, manganese(III) ion, bromate or iodate. U.S. patent application Ser. No. 337,620 does not teach or suggest any metal ion other than copper(II) as the oxidant to eliminate ascorbate interference.
As will be demonstrated more fully hereinafter, it has been found that a cooxidant selected from the group consisting of bromate ion, chlorate ion, perchlorate ion, chromate ion, organic oxidants like a peroxide, a hydroperoxide or a N-halo compound, and combinations thereof, when included in an indicator reagent composition of the present invention having an essentially neutral pH, oxidizes the reduced form of a metal ion, said metal ion included in the composition to eliminate primary ascorbate interference, back to the oxidized form of the metal ion. Such a result is both surprising and important. The result is surprising because of the pH range wherein the cooxidant, like bromate ion or chromate ion, oxidizes the metal; and the result is important because the metal ion, originally included in the composition to eliminate primary ascorbate interference, itself becomes a secondary interferent when the reduced form of the metal ion interacts with the colored, oxidized form of the indicator dye to reduce the dye to its colorless, reduced form. Accordingly, because of this secondary interference, the indicator dye apparently does not undergo a full color transition in response to the concentration of the predetermined analyte in the test sample. Therefore, an erroneously low assay result for the predetermined analyte is provided.
However, the composition, method and device of the present invention provide an accurate assay for a predetermined analyte that is capable of interacting with an oxidase enzyme or that exhibits peroxidase activity. Surprisingly, the method and composition of the present invention essentially eliminate both the primary and the secondary interferences attributed to ascorbate present in the test sample. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase test strip assay of blood, urine and other test samples for a predetermined analyte by utilizing an indicator reagent composition that includes a metal ion complex to eliminate primary ascorbate interference and a cooxidant selected from the group consisting of bromate ion, chlorate ion, perchlorate ion, chromate ion, organic oxidants like a peroxide, a hydroperoxide or a N-halo compound, and combinations thereof to eliminate secondary interferences attributed to the reduced form of the metal ion of the metal ion complex.